Available at w w w.vet er inar yw or ld.or g/ Vol.9/ July- 2016/ 7.pdf Ope n Acce ss
A com pa r ison of t it e r s of a n t i- Br u ce lla a n t ibodie s of n a t u r a lly in fe ct e d
a n d h e a lt h y v a ccin a t e d ca t t le by st a n da r d t u be a gglu t in a t ion t e st ,
m icr ot it e r pla t e a gglu t in a t ion t e st , in dir e ct h e m a gglu t in a t ion a ssa y , a n d
in dir e ct e n z y m e - lin k e d im m u n osor be n t a ssa y
Anj u Mohan1, Har i Mohan Saxena1 and Puneet Malhot ra2
1. Depar t m ent of Vet er inar y Micr obiology, College of Vet er inar y Science, Gur u Angad Dev Vet er inar y and Anim al Sciences Univer sit y, Ludhiana - 141 004, Punj ab, I ndia; 2. Depar t m ent of Anim al Genet ics and Br eeding, College of Vet er inar y
Science, Gur u Angad Dev Vet er inar y and Anim al Sciences Univer sit y, Ludhiana - 141 004, Punj ab, I ndia.
Cor r e spon din g a u t h or : Har i Mohan Saxena, e- m ail: hm saxena@yahoo.com ,
AM: anz089@gm ail.com , PM: dr.puneet m alhot ra@r ediffm ail.com
Re ce iv e d: 07- 04- 2016, Acce pt e d: 08- 06- 2016, Pu blish e d on lin e : 12- 07- 2016
doi: 10.14202/ vet w or ld.2016.717- 722 H ow t o cit e t h is a r t icle : Mohan A, Saxena HM, Malhot ra P ( 2016) A com par ison of
t it er s of ant i- Br ucella ant ibodies of nat urally infect ed and healt hy vaccinat ed cat t le by st andar d t ube agglut inat ion t est , m icr ot it er plat e agglut inat ion t est , indir ect hem agglut inat ion assay, and indir ect enzym e- linked im m unosor bent assay, Vet er inar y Wor ld, 9( 7) : 717- 722.
Abst r a ct
Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19.
Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT), microtiter plate agglutination test (MAT), indirect hemagglutination assay (IHA), and indirect enzyme-linked immunosorbent assay (iELISA) as per standard protocols.
Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001). The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005). The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002). However, the difference in mean iELISA titers of infected cattle (1.3678±0.014) and healthy vaccinated cattle (1.367±0.014) was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms.
Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.
Keywords: antibody titers, Brucella, brucellosis, Brucella abortus S19 vaccine, bovine brucellosis.
I n t r odu ct ion
Brucellosis is a major bacterial zoonosis of global importance. Brucellosis occurs worldwide but is much controlled in developed countries by routine screening of domestic animals and vaccination program. Clinical disease is still common in Middle East, Asia, Africa, South and Central America, the Mediterranean Basin, and the Caribbean. About 500,000 cases of human bru-cellosis are estimated to occur worldwide every year. It causes heavy economic loss to the animal industry through abortion, delayed conception, and temporary or permanent infertility in the affected animals [1].
Bovine brucellosis is endemic in all states of India. In India, the occurrence of brucellosis is to the extent of 10% in the marginal herds and 50% in organized farms, and the socio-economic impact of the disease was estimated to run over Rs. 500 crores annually. In Punjab, overall 17.7% prevalence of brucellosis was reported in cattle and buffaloes [2,3]. Brucellosis in animals is clinically characterized by late-term abor-tions and retention of placenta in females and orchitis and epididymitis in males, with excretion of organ-isms in semen, uterine discharges, and in milk [4]. Once infected, the animal may continue to shed bacte-ria and remains a source of infection to others for long period [3]. Mass vaccination is crucial for the control and eradication of bovine brucellosis. The widely used vaccine against brucellosis is derived from the smooth live vaccine strain (S19) for cattle. Although it has some undesirable traits, it has proven to be very useful under most conditions [5-7]. Brucella abortus S19 has been effective for the control of brucellosis
in adult bovines and preventing abortion as well as decreasing the prevalence in herds [8].
The antibodies induced by vaccination interfere in serological diagnosis of brucellosis. Little data are available in the published literature on comparison of antibody titers due to vaccination and those due to nat-ural infection in cattle. The present study was, there-fore, undertaken to explore this aspect of serology of bovine brucellosis.
M a t e r ia ls a n d M e t h ods
Et h ica l a ppr ov a l
All the experimental protocols performed on cattle were approved by the Institutional Animal Ethics Committee (IAEC). Animals were kept in IAEC approved facilities and received feed and water ad libitum.
I n fe ct e d a n d v a ccin a t e d ca t t le
A total of 15 naturally infected brucellosis pos-itive adult cattle, which were vaccinated during calf hood with B. abortus strain 19 vaccine (Bruvax; Indian Immunologicals), and 6 normal healthy calf hood vaccinated Holstein-Friesian crossbred adult cattle maintained at the University Dairy Farm were included in the study.
B. a bor t u s st r a in 1 9 ( v a ccin e st r a in )
The standard vaccine strain B. abortus strain 19, procured from the Biological Standardization Division, IVRI, Izatnagar, was used in the present study.
Colle ct ion of se r u m
Blood samples were collected from cattle through jugular vein for obtaining sera for studying the humoral immune response of the animals. Sera were separated and stored at −20°C until further use. An a ly sis of im m u n e r e spon se s
Rose Bengal plate agglutination test (RBPT)
Equal volumes (10 μl each) of RBPT colored anti-gen (Punjab Veterinary Vaccine Institute, Ludhiana) and test serum were mixed on a clean glass slide [9] with the help of a sterilized toothpick. The slide was observed for 4 min for the formation of clumps. The
formation of clumps was considered a positive test, whereas the absence of clear clumps was considered a negative reaction.
Est im a t ion of a n t ibody t it e r s by st a n da r d t u be a gglu -t in a -t ion -t e s-t ( STAT)
The standard OIE method [10] was followed (Table-1). The highest serum dilution showing 50% agglutination was taken as the end point for the titer. A titer of 1:40 or above was considered positive. Controls
1: Tube No. 08 - 25% agglutination 2: Tube No. 09 - 50% agglutination 3: Tube No. 10 - 75% agglutination. M icr ot it e r pla t e a gglu t in a t ion t e st ( M AT)
MAT was performed as per the method reported earlier [11].
Procedure
a. Serum samples were serially diluted two-fold in a final volume of 100 μl in 96 well U-bottom microtiter plate (Tarsons)
b. Equal volume of 100 μl B. abortus plain antigen (Punjab Veterinary Vaccine Institute, Ludhiana) was added to each well. Negative control well containing 100 μl of sterilized normal saline solu-tion (NSS) and 100 μl of the antigen was also kept c. The plate was covered with a lid and incubated at
37°C for 24 h followed by incubation at 4°C for 1 h d. The formation of matt signified agglutination
while button formation was indicative of a neg-ative reaction. Titers (log10 values) were recorded as the reciprocal of the highest dilution of the serum giving at least 50% agglutination.
I n dir e ct h e m a gglu t in a t ion a ssa y ( I H A)
The method reported earlier [12] was followed with minor modifications.
Fixation of sheep red blood cells (sRBCs)
Sheep blood was collected aseptically by jug-ular vein puncture into Alsever’s solution (1:1) and kept at 4°C for 7 days before further processing. The
Ta ble - 1 : STAT pr ot ocol.
Tu be N o. Ca r bol sa lin e ( in m l)
Te st se r u m ( in m l) B. a bor t u s pla in
a n t ige n ( in m l)
Fin a l dilu t ion
1 0.8 0.2 0.5 1: 10
2 0.5 Ser ial dilut ion was per for m ed aft er t hor ough m ixing. 0.5 m l of t he cont ent s was t ransfer r ed fr om t ube No. 1 t o t he next t ube up t o t ube No. 7. Finally, 0.5 m l of t he cont ent s was discar ded fr om t ube No. 7
0.5 1: 20
3 0.5 0.5 1: 40
4 0.5 0.5 1: 80
5 0.5 0.5 1: 160
6 0.5 0.5 1: 320
7 0.5 0.5 1: 640
8 1.25 0.75
9 1.50 0.50
10 1.75 0.25
blood was then centrifuged at 1500 rpm for 10 min to pack the erythrocytes. The packed RBCs were washed three times with 5-6 volumes of chilled NSS by cen-trifugation. Finally, a 10% (v/v) suspension of RBCs was prepared in chilled NSS and stored at 4°C. Fixation and treatment of sRBCs with tannic acid a. A 1% v/v solution of glutaraldehyde was prepared
in NSS and stored at 4°C. Equal volumes of chilled glutaraldehyde solution and 10% washed sRBC suspension were mixed and allowed to stand at 4°C for 30 min with intermittent gentle stirring. b. The sensitized sRBCs were packed by
centrifu-gation at 1500 rpm for 10 min at room tempera-ture followed by three washes in NSS to remove free glutaraldehyde and resuspended in the same buffer containing 0.1% sodium azide to yield a 10% suspension of sRBCs. The glutaraldehyde fixed sRBCs (G-sRBCs) were then stored at 4°C. c. A 10% suspension of G-sRBCs was mixed with
an equal volume of phosphate-buffered saline (PBS) containing 0.005% tannic acid (w/v) and incubated at 37°C with occasional shaking. The tanned G-sRBCs (TG-sRBCs) were pelleted by centrifugation at 650 ×g for 10 min at room tem-perature and washed three times with PBS to yield a 10% suspension.
Pr e pa r a t ion of a n t ige n
The antigen prepared as described earlier was heated at 56°C for 30 min in a water bath with frequent shaking. Heat treated suspension was then centrifuged at 8000 rpm for 15 min at 4°C. The clear supernatant was separated and stored at −20°C until use.
Sensitization of TG-sRBCs with antigen
a. One volume of packed RBCs and 15 volumes of the antigen were mixed and incubated for 1-2 h at 37°C in a water bath with frequent shaking
b. The sensitized cells thus prepared were washed 3 times with NSS by centrifugation at 2000 rpm for 5 min. After the final wash, packed cells were resus-pended in chilled NSS to obtain 1% suspension. Adsorption of serum samples
a. To remove the heterophile antibodies, all the test serum samples (3 volumes) were adsorbed with packed sRBCs (1 volume) for 2 h at 37°C with periodic shaking before the test proper. The RBCs were removed by centrifugation
b. The suspension was centrifuged at 600 ×g for 15 min at 4°C in a refrigerated centrifuge. The supernatant was collected and used for the test. Test protocol for IHA
a. PBS (160 μl) and inactivated adsorbed serum (40 μl) were added to the first well (1 in 4 dilu-tion), and 100 μl of PBS was added to all the other wells of a 96 well U-bottom microtiter plate (Tarsons). Two-fold serial dilutions of serum were made in a final volume of 100 μl
b. An equal volume (100 μl) of the 0.5% sensitized RBC suspension was added to all the wells. The plates were shaken and left at room temperature for 2 h.
Coarse agglutination of RBCs (matt formation) indicated a positive result and formation of a small button of deposited cells was considered as a negative result.
Controls
The following three controls were included with each test:
• Antigen control: 100 μl of sensitized and adsorbed RBCs
• RBC control: 100 μl of 1:4 dilution of serum and 100 μl of sensitized RBCs
• Serum control: 100 μl of untreated erythrocytes and 100 μl of test sera.
En z y m e - lin k e d im m u n osor be n t a ssa y ( ELI SA)
The serum samples of cattle were tested using Brucellosis Serum ELISA test kit (Idexx). The kit is based on indirect ELISA (iELISA) using inactivated antigen of B. abortus. The binding of the antibod-ies in cattle serum samples with precoated inacti-vated antigen on microtiter plate is detected by per-oxidase-labeled anti-ruminant immunoglobulin G (IgG). The degree of the color that develops (optical density [OD] measured at 450 nm) is directly pro-portional to the amount of antibody specific for B. abortus present in the sample. The diagnostic rele-vance of the result is obtained by comparing the OD in wells containing the samples with the OD from wells containing the positive control. Antibody titers were calculated using an equation of regression. Procedure
All reagents were thawed to 25°C and mixed by gentle vortexing before use.
a. Dispensed 90 μl of diluted wash solution (1:10) into each well of the microtiter plate
b. Added 10 μl of the undiluted serum samples and controls into the appropriate wells of the microti-ter plate making the final dilution 1:10
c. Mixed the contents within each well by gently shaking the microtiter plate
d. Covered the microtiter plate with a lid and incu-bated for 60 min at 37°C in a humid chamber e. Washed each well with approximately 300 μl
wash solution 3 times. Aspirated liquid contents of all the wells after each wash. Following the final aspiration, firmly tapped the residual wash fluid from each plate onto absorbent material. Drying of plate between washes and before the addition of the next reagent was avoided
f. Dispensed 100 μl conjugate into each well g. Covered and incubated the microtiter plate for
60 min at 37°C in a humid chamber
i. Dispensed 100 μl of 3,3’,5,5’-Tetramethylbenzidine substrate into each well and incubated the sub-strate at 18-26°C for 15 min
j. Stopped the color reaction by adding 100 μl of stop solution per well
k. The OD was recorded in an ELISA reader at a wavelength of 450 nm.
Ca lcu la t ion of a n t ibody t it e r s
Antibody titer (Log10) Y= a + bx
Where, constant a=1.35; constant b=0.05; X=OD value of a test well/Mean +3 standard deviation value of negative control wells. The standard error of the Y estimate (antibody titer) was + 0.19 log10.
St a t ist ica l a n a ly sis of da t a
Data pertaining to antibody titers by STAT, MAT, IHA, and iELISA were statistically analyzed by analy-sis of variance and t-test.
Re su lt s a n d D iscu ssion
Antibody titers of infected or vaccinated cat-tle were estimated by STAT, MAT, IHA, and iELISA (Tables-2 and 3, Figure-1). STAT revealed the mean titers in infected cattle to be 1.963±0.345, and the cor-responding values in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p<0.0001). The mean MAT titers in infected cattle were 2.244±0.727, and the corresponding values in healthy vaccinated cattle were 1.200±0.155. The difference was very significant (p<0.005). IHA revealed the mean titers in infected cattle to be 2.284±0.574, and the correspond-ing values in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002). Interestingly, the difference between the mean iELISA titers of infected cattle (1.3678±0.014) and healthy vac-cinated cattle (1.367±0.014) was non-significant.
Even though a number of antigenic components of Brucella have been characterized, the antigen that
dominates the antibody response is the lipopolysac-charide (LPS). Numerous outer and inner membrane, cytoplasmic, and periplasmic protein antigens have also been characterized. Some are recognized by the immune system during infection and are potentially useful in diagnostic tests. The L7/L12 ribosomal proteins are important in stimulating cell-mediated responses [13].
Immune response of host to Brucella infection is mediated through both humoral and cell-mediated immunity [14]. The role of humoral immunity against intracellular bacterial infections is limited and not pro-tective. Antibody-mediated opsonization by Igs (IgM, IgG1, IgG2a, and IgG3) enhances phagocytic uptake of bacteria, limiting the level of initial infection with Brucella but has little effect on the intracellular course of Brucella infection [15,16].
B. abortus strain 19 is used as a live vaccine and is normally given to female calves aged between 3 and 6 months as a single subcutaneous dose of 5-8 × 1010 viable organisms. It is believed to induce protective
Ta ble - 2 : Ant ibody t it er s of cat t le nat urally infect ed w it h br ucellosis.
S. N o. An t ibody t it e r s ( log1 0)
STAT M AT I H A iELI SA
1 1.903 2.204 2.204 1.4259
2 2.204 3.107 3.408 1.4328
3 1.602 3.408 2.505 1.4296
4 1.903 3.107 3.107 1.4264
5 1.602 1.602 1.602 1.3615
6 2.204 1.602 1.903 1.4226
7 2.806 3.709 3.107 1.4243
8 1.903 2.204 2.505 1.4251
9 1.602 1.903 1.602 1.3878
10 2.204 1.602 2.505 1.4539
11 1.602 1.903 1.903 0.6101
12 1.602 1.602 1.602 1.2935
13 2.204 1.602 1.903 0.4963
14 1.903 1.903 2.204 1.4084
15 2.204 2.204 2.204 1.4732
Mean± SD 1.963± 0.345 2.244± 0.727 2.284± 0.574 1.3678± 0.014
SD= St andar d deviat ion, STAT= St andar d t ube agglut inat ion t est , MAT= Micr ot it er plat e agglut inat ion t est , I HA= I ndir ect hem agglut inat ion assay t est , iELI SA= I ndir ect enzym e- linked im m unosor bent assay
Figu r e - 1 : Ant ibody t it er s in br ucellosis infect ed or vaccinat ed
immunity against B. abortus in cattle [17]. However, the effectiveness of this vaccine varies with the age of vaccination, dose, route, and prevalence of brucellosis in the herd [18]. B. abortus strain 19 vaccine remains a reference vaccine to which any other vaccine is compared. Persistent antibodies could be detected up to 10-11 months post-vaccination [19].
As evident from our present data, infected ani-mals have very high titers of antibodies compared to the vaccinated animals. However, the high titers do not indicate whether these are protective antibodies due to vaccine or acute response to infection in the absence of a differentiation of infected from vacci-nated animals (DIVA) assay or whether they are of any relevance to prognosis. Since the infected animals were the ones who had already been vaccinated during calf hood, the infection in these animals may suggest that the vaccine was unable to induce protective levels of antibody. Second, the heightened antibody response after infection may indicate a secondary immune response to the genus-specific antigens of Brucella.
The different titers observed in the same ani-mals by agglutination assays (STAT, MAT, and IHA) and iELISA can be reconciled with the fact that these assays target antigens of different nature, i.e., aggluti-nation assays are directed toward particulate antigens, whereas ELISA detects immune response to soluble antigens. ELISA is generally used to detect IgG anti-bodies [20]. In brucellosis, specific IgM antianti-bodies dominate during the acute phase of the disease [21]. Specific IgG antibodies are present in the serum of patients at later stages of the illness and in the serum of relapsing patients [22]. ELISA is used to discriminate between the presence of specific IgM and IgG anti-bodies and to roughly assess the stage of illness [23].
In many countries, STAT is the routine diagnos-tic test for human and animal brucellosis. It has been reported [24] that STAT has a greater accuracy than that of the RBPT (93.3% and 76.6%, respectively). In a study [25], Brucella antibodies were investigated in bovine sera by RBPT, serum agglutination test, MAT, and 2-mercaptoethanol MAT, and MAT was deter-mined as a fast, reliable, and economic test. On evalu-ation of canine brucellosis by MAT, it was shown [26] that MAT was more sensitive, simpler to perform, and easier than tube agglutination test.
A study [27] has shown that the use of sheep erythrocytes sensitized with a specific LPS antigen in the IHA test provided a specific method, which is more sensitive than the agglutination test. A study [28] was carried out to compare the efficacy of RBPT, STAT, and DotELISA in immunological detection of antibodies to B. abortus in sera. The study revealed that DotELISA was the most sensitive of the three tests used. In a study by Ghodasara et al. [29], STAT and iELISA were compared for detection of Brucella antibodies in cows and buffaloes. The seropositiv-ity was found highest by iELISA (25%) followed by STAT (14.45%). iELISA, RBPT, MAT, and PCR were evaluated [30] for diagnosis of brucellosis in buffa-loes, and it was concluded that iELISA detected more samples as positive among these tests. Currently, no DIVA vaccine against brucellosis is available in the market. Identification of extracellular proteins from Brucella may aid in discovery of better vaccines or diagnostic molecules [31].
Since cell-mediated immunity is known to play an important role in brucellosis, it would be perti-nent to incorporate antigens and adjuvants in the vaccine which could generate cellular immunity of a high protective level to be effective in control of brucellosis.
Con clu sion
We determined the antibody titers by STAT, MAT, IHA, and ELISA in cattle naturally infected with brucellosis and normal healthy adult cattle vac-cinated during calf hood with strain 19. The differ-ences between the mean STAT, MAT, and IHA titers of infected cattle and healthy vaccinated cattle were highly significant (p<0.0001, p<0.005, and p=0.0002, respectively). However, the difference in mean iEL-ISA titers of infected cattle and healthy vaccinated cat-tle was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.
Au t h or s’ Con t r ibu t ion s
AM did the experiments; HMS conceived the idea, planned the study, and wrote manuscript; PM maintained the animals in the farm. Both authors read and approved the final manuscript.
Ta ble - 3 : Ant ibody t it er s of healt hy cat t le vaccinat ed w it h B. abor t us st rain 19 vaccine.
S. N o. An t ibody t it e r s ( log1 0)
STAT M AT I H A iELI SA
1 1.301 1.301 1.301 1.361
2 1.301 1.301 1.301 1.361
3 1.301 1.301 1.301 1.361
4 1.000 1.000 1.000 1.361
5 1.000 1.000 1.000 1.367
6 1.301 1.301 1.301 1.396
Mean± SD 1.200± 0.155 1.200± 0.155 1.200± 0.155 1.367± 0.014
Ack n ow le dgm e n t
Dr. Parkash Singh Brar, Director, Livestock Farms, Guru Angad Dev Veterinary and Animal Sciences Universitypermitted us to carry out the study on cattle maintained in the University Dairy Farm and Dr. Mayank Rawat, Principal Scientist, Indian Veterinary Research Institute discussed with Hari Mohan Saxena about post vaccinal antibody titers in case of S-19 vaccine.
Com pe t in g I n t e r e st
The authors declare that they have no competing interests.
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